A large number of specific cellular proteins can be translocated across distinct cellular membranes. Targeting of these proteins is specified by membrane specific signal sequences. It is not known how, after targeting, the polypeptide crosses the membrane. We have studied the permeability properties of membranes fractions enriched in translocation activity by fusing such vesicles to planar phospholipid membranes. We find channel activity, with unitary conductances of 20, 55, 80, and 115 pS in 45 mM Kglutamate. A similar large channel was seen in both dog pancreatic microsomes and E. Coli inverted vesicles. Since these two membranes translocate proteins, and glutamate is an amino acid, it is possible that protein translocation occurs through these structures. We have continued to use our newly-developed technique of transport-selection to study the gap junction of rat liver. It is generally believed that proteins called connexins form gap junction channels through which neighboring cells exchange cytoplasmic factors. We firmly establish that connexin 32 forms ion channels in single phospholipid membranes, by functional purification and monoclonal antibody identification of the channel-forming protein. Connexin 32 channels show heterogeneous conductances, weak cation selectivity, and asymmetric voltage sensitivity, properties consistent with those expected for a structure which composes one-half the intracellular gap junction channel.